Dinuclear Fe centers are found in a variety of proteins with diverse activities. Our interest is focussed on methane monooxygenase, which activates dioxygen for incorporation into a variety of substrates, including methanol. XAS studies during the previous proposal focused on structural characterization of the oxidized, semi-met and reduced states of the hydroxylase component, which contains the diiron active site. This proposal is for the continuation of our XAS studies of MMO. In particular we propose to i) study protein-substrate interactions in the presence/absence of other components of the MMO system (B and C). We will also utilize Br-labeled substrates using Br K-edge XAS measurements; ii) study specifically modified components which have engineered changes in specific amino acid residues; iii) investigate specific intermediates in the reaction pathway using time-resolved experiments with freeze-quench methodology; and iv) extend our understanding of edges of such diiron centers to gain further insights into the electronic structure of the diiron site. Through such studies, we hope to gain further insights into the details of the structure and function of this important enzyme system.